Difference between revisions of "Team:Potsdam/notebook/lowcopy"

Line 1,806: Line 1,806:
 
<div style="text-align: justify; margin-left:20px">
 
<div style="text-align: justify; margin-left:20px">
 
- plated on cap plates (200 µl and rest) </div>
 
- plated on cap plates (200 µl and rest) </div>
 +
<br> <br>
 +
 +
<b> Tuesday, 09/19/17 </b>
 +
<br> <br>
 +
<div style="text-align: justify;">
 +
- Colony PCR of dCas9-LacI in pSB1C3 <br>
 +
- mastermix for 10 clones + negative control </div>
 +
<br> <br>
 +
<table>
 +
  <tr>
 +
    <th  width="50%" align="center"><b>component</b></th>
 +
    <th  width="50%" align="center"><b>volume in µl</b></th>
 +
  </tr>
 +
  <tr>
 +
    <td align="center">water</td>
 +
    <td align="center">100,8</td>
 +
  </tr>
 +
  <tr>
 +
    <td align="center">Q17</td>
 +
    <td align="center">9,6</td>
 +
    <td align="center">-> annealing Tm 52°C, binds to backbone</td>
 +
  </tr>
 +
  <tr>
 +
    <td align="center">Q20</td>
 +
    <td align="center">9,6</td>
 +
    <td align="center">-> annealing Tm 52,6°C, binds to dCas9</td>
 +
  </tr>
 +
  <tr>
 +
    <td align="center">Red Taq</td>
 +
    <td align="center">120</td>
 +
  </tr>
 +
</table>
 +
<br> <br>
 +
<div style="text-align: justify;">
 +
- fragment length: 1,6 kb -> elongation time 23 sek <br>
 +
- gel picture: except for clone 3 and 9 all clones positive<br>
 +
- inoculated miniprep cultures (5 ml LB + 5 µl CAP) of clone 4,7,8 and 10<br>
 +
- Colony PCR of P4<br>
 +
- mastermix for 10 clones + negative control </div>
 +
<br> <br>
 +
<div style="text-align: justify;">
 +
- Colony PCR of dCas9-LacI in pSB1C3 <br>
 +
- mastermix for 10 clones + negative control </div>
 +
<br> <br>
 +
<table>
 +
  <tr>
 +
    <th  width="50%" align="center"><b>component</b></th>
 +
    <th  width="50%" align="center"><b>volume in µl</b></th>
 +
  </tr>
 +
  <tr>
 +
    <td align="center">water</td>
 +
    <td align="center">100,8</td>
 +
  </tr>
 +
  <tr>
 +
    <td align="center">Q18</td>
 +
    <td align="center">9,6</td>
 +
  </tr>
 +
  <tr>
 +
    <td align="center">Q24</td>
 +
    <td align="center">9,6</td>
 +
  </tr>
 +
  <tr>
 +
    <td align="center">Red Taq</td>
 +
    <td align="center">120</td>
 +
  </tr>
 +
</table>
 +
<br> <br>
 +
<div style="text-align: justify;">
 +
- annealing Tm 52°C <br>
 +
- fragment length: 700 bp -> elongation time: 10,5 sek<br>
 +
- gel picture: no bands! <br>
 +
- possible reasons: not enough DNA (IAAH-PP7 with corrected overhangs) applied due to low concentration (see 18.09.)
 +
or colony PCR did not work properly. In case of that miniprep cultures were inoculated with clone 1,6,8 and 10 (10 ml LB + 10 µl Amp)<br>
 +
- Tomorrow test digest of those!<br>
 +
- master plate with cellulose membrane in 37 °C incubator<br>
 +
- Amplification of IAAH-PP7 with corrected overhangs<br>
 +
- gradient PCR: tried 1:10 and 1:100 dilutions<br>
 +
- master mix for 11 x 10 µl aliquots </div>
 +
<br> <br>
 +
<table>
 +
  <tr>
 +
    <th  width="50%" align="center"><b>component</b></th>
 +
    <th  width="50%" align="center"><b>volume in µl</b></th>
 +
  </tr>
 +
  <tr>
 +
    <td align="center">water</td>
 +
    <td align="center">48</td>
 +
  </tr>
 +
  <tr>
 +
    <td align="center">Q39</td>
 +
    <td align="center">8</td>
 +
  </tr>
 +
  <tr>
 +
    <td align="center">Q26</td>
 +
    <td align="center">8</td>
 +
  </tr>
 +
  <tr>
 +
    <td align="center">Q5 master mix</td>
 +
    <td align="center">80</td>
 +
  </tr>
 +
</table>
 +
<br> <br>
 +
<div style="text-align: justify;">
 +
- per tube: 9 µl master mix + 1 µl template (1:10, 1:100 or 1:1000)<br>
 +
- same gradient, same conditions <br>
 +
- gel: 1% agarose; 150 V, 30 min<br>
 +
- clear bands visible, all clones positive!<br>
 +
- annealing temperatures between 65-67 °C (all three equaly good) and concentration of 1:1000 <br>( 0,01 ng/µl) worked best<br>
 +
- extracted all bands at 1,8 kb from gel -> three yellow labeled tubes in the fridge</div>
 
<br> <br>
 
<br> <br>
 
</p>
 
</p>

Revision as of 20:13, 1 November 2017

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Lab book

We splitted our labbook into the following parts:



Friday, 07/21/17

- took pSB4A5 backbone out of DNA distribution Kit and transformed into E.coli
(rest from Kit was put in -20 °C)

Tuesday, 07/25/17

- Inoculated LB media with psB4A5

Wednesday, 07/26/17

- Miniprep with Promega “Plus SV Miniprep DNA Purification System”
- 2 approaches with psB4A5
- final concentration:
- approach 1: 30 ng/µl
- approach 2: 40 ng/µl


Thursday, 08/10/17

- glycerol stock (30%) of IG_C_P1_G (pSB4A5)
- prepared 10 mL preculture with pdCas9-Bacteria E.coli from plate from Fabian for Miniprep


Monday, 08/14/17

- primer resuspended to 100 uM (except Q8):

primer volume in µl
Q5 354
Q6 957
Q7 749
Q8 657 (83,1 uM)
Q15 956
Q16 1455
Q17 1693
Q18 1440


- diluted all primers to 10 uM in separate aliquotes
- prepareed 3 separate PCRs with primer pairs:
Q5/Q8 (Eppi A), Q6/Q7 (Eppi B), Q15/Q16 (Eppi c)
- pipetting scheme:
Q5 2X MasterMix: 5 µL
10 uM Primer: each 0,5 µL
Template: 1 µL of 1:100 diluted Miniprep (about 0,5 ng)
ddH2O: 3 µL
total: 10 µL
- cycling conditions (prog01 in Epepndorf Mastercycler nexus gradient (Torsten):

temperature time in sec
98 °C 30
98 °C 10
64 °C 20
72 °C 110
72 °C 120


- after PCR: gel electrophoresis as follows:
- 2.5 µL DNA + 2µL Loading Dye + 7.5 µL (total 12 µL)
- ran gel at 120 V for 40 minutes
gel picture:

- order of pockets: ABC, A and B are correct size, C did not work well, there is a band at the desired result and one at the unopened plasmid (and several other stuff) so PCR has to be done again
- dCas9 fragments look good and can be worked with tomorrow


Tuesday, 08/15/17

1. DpnI digestion of dCas9 fragments A + B

component volume in µl
DpnI 2,6
DNA 6,5
cutsmart-Buffer 5,0
H2O 35,9
final volume 50


- Duration: 1 hour

IMPORTANT: Fabian told me you can just add about 1 µL DpnI to the reaction-mix

2. Gel run of dCas9 A + B
- let it run on gel for purification
- 140 V a 40 min
- cut out was done by Natalie and Pauline

3. Linearization PCR of psB4A5 (second atempt) via gradient PCR
- made new PCR using Q15/Q16 primers and psB4A5 plasmid fpr linearization
- used 3 different DNA concentrations and 3 different annealing temperatures (9 reactions total)


component 1 reaction:
volume in µl		 10 reaction MM:
volume in µl
HiQ 5 50
primer 1,25 12,5
DNA variable (1 µl) variable
H2O 17,5 175
final volume 25 250


- annealing temperatures: A = 60°C/B = 64,5°C/C = 68°C
- DNA concentrations: 0,1 = 1:1000 / 1 = 1:100 / 10 = 1:10
Scheme:

0,1 A 0,1 B 0,1 C
1 A 1 B 1 C
10 A 10 B 10 C


Wednesday, 08/16/17

1. Gel run of psB4A5 gradient PCR (A,B,C,0.1,1,10)
- run at 120 V a 40 min -> only 2 bands at well 1A
- run at 120 V another 30 min -> lost DNA from gel
- 1A (60°C/1:100 DNA) might have worked so I did a PCR purification for this one
2. Gel purification of dCAs9 A + B fragments and 1A-psB4A5 linearized plasmid
- used Promega PCR/Gel purification kit: final concentration pSB4A5 18 ng/µL
https://www.promega.de/resources/protocols/technical-bulletins/101/wizard-sv-gel-and-pcr-cleanup-system-protocol/
IMPORTANT: We use AE-buffer for elution in the last step (50 µL) instead of water!
3. Fusion PCR of dCAs9 fragments A + B
- Primer used: Q5 + Q6


component volume in µl
Q5-MM 12,5
DNA 2(1 pro fragment)
H2O 10,5
final volume 25


IMPORTANT: Fusion PCR done like team munich!
https://static.igem.org/mediawiki/2014/c/c9/LMU_Munich14_FusionPCR.pdf
- first made a PCR mix of 25 µL without primers
- then run mix in thermocycler with programm (fusion pcr)
- in the middle there is a hold step in the programm
- you then add the primers mixed with buffer (5 µL total volume) for an endvolume of 30 µL
- IG_C_Q19/20 and Q24 resuspended auf 100 µM and diluted to 10 µM


Thursday, 08/17/17

1. dCas9:
- DpnI- digested Fusion-PCR product (1 µL in PCR-tube for 1h at 37 °C)
- ran 0,7 % agarose gel --> negative result
- gel picture:



- possible reasons: not enough DNA in PCRPCR cleanup was done and not gel extraction, wrong PCR protocol
- PCR with Q5-Q8 was done again (see above for protocol)
- no test gel but preperative gel and gel extraction with Promega protocol
- dCas9-A fragment: 2,8 ng/µL, dCas9-B fragment: 3,3 ng/µL
- not enough DNA again for PCR, tomorrow run PCR with 50 µL reaction tomorrow!


2. pSB4A5:
DpnI-digestion:
- 1 µl added to the PCR mix and incubated 1 h at 37 °C
- SLiCE reaction of pSB4A5 with IAAM-MS2 + IAAH-PP7:
because of to low DNA concentration we needed to increase the concentration by drying the DNA
- for this reaction we take:
2,8 µl backbone (50 ng, 3395 bp, 18 ng/µl)
+ 32,2 µl IAAM-MS2 (1:10 ratio, 2188 bp, 10 ng/µl)
+ 26,4 µl IAAH-PP7 (1:10 ratio, 1790 bp, 10 ng/µl)
all in one eppi + 38,6 µl ddwater to get 100 µl
to the 100 µl we added:
+ 1/10 of original volume (100µl) 3 M Sodium acetate, pH 5,2 --> 10 µl
+ 2 x original volume 100% ethanol (needs to be cooled down for 20 min at -20°C) --> 200 µl
mix thorougly
- put 30 min to -20 °C (in this time cool down centrifuge to 4 °C)
- centrifuge 10 min with maiximal speed at 4 °C
- discard supernatant with a pipette, BE CAREFUL!!!, pellet is maybe (in most cases, because we don't have much DNA) nit visible, pay attenteion on the orinetation of the Eppis in the centrifuge (lids to the outside) and go with the pipette onto this side of the Eppi, where the pellet DON'T is, DON'T TOUCH THE PELLET!!!
- add 300 µl 70 % ethanol (needs to be cooled down for at least 20 min at -20 °C)
- centrifuge 10 min with maiximal speed at 4 °C
- again CAREFULLY discard supernatant with a pipette
- make a short spin 10-30 sec (Short-Button) and take carefully additional supernatant with a pipette
- put Eppi open lid for 10 min to 37 °C
- add SLiCE reaction directly to this Eppi (NO RESUSPENSION BEFORE)
SLiCE reaction:
Eppi with dried backbone, IAAM-MS2 and IAAH-PP7
+ 1 µl 10 x SLiCE buffer
+ 1 µl PPY SLiCE extract
+ 8 µl ddwater
--> 10 µl SLiCE reaction
- incubate 1 h at 37°C, no shaking (flip a few times to the Eppi to resuspend the DNA)
- Transformation in E.coli clls with all the 10 µl SLiCE reaction in the same Eppi like the transformation protocol, no changes from protocol
- 200 µl to Ampicillin-LB-agar plates
- and centrifuge rest of the culture, discard most of the supernatant, resuspend in the rest supernatant and put this resuspenden rest also to an Ampicillin-LB-agar plate
- plates over night to 37 °C
4. for tomorrow:
Colony-PCR with the SLiCE plate and at the same time preparation of the Slakowski-Assay plate

Friday, 08/18/17

- no colonies on pSB4A5 plate
- did q5-Q8 PCr again with 50 uL reaction following protocol (5 uL of 1:100 diluted miniprep as template)
used Q5678 protocol in Torsten Thermocycler
- gel extraction:
fragment A: 3,7 ng/uL
fragment b: 31,4 ng/uL
- PCR Q5/Q8 again for fragment A with gradient PCR
- for Monday: program in Torsten: "Q58":
- use 4 different wells/temperatures:
- well 1: 60°C; 5: 62,5°C; 7:64,5 °C; 9: 66,5°C
- elongation temperature reduced to 30 sec
- make 50 uL reaction again,
afterwards: gel extraction and fusion PCR of dCas-A and dCas9-B
ask Fabian for better Fusion-PCR cycler program (the one from last time was unnecessarily long according to Fabian)


Monday, 08/21/17

- gradient PCR for dCas9-A fragment
- Mastermix for 10 reactions (10 µl/ reaction):


component volume in µl
Q5 2xMastermix 50
primer Q5 (10µM) 5
primer Q8 (10µM) 5
water 20
template (~1ng) 20
total volume 100


- template: pdCas9 from miniprep (48,3 ng/µl) -> dilution: 1:100 ~ 0,5 ng/µl
- PCR program: dcas9a_gradient (in Torsten) (also changed elongation time to 30 s)
temperatures of gradient:
60°C 60,2°C 61,5°C 62,5°C 64,5°C 65,5°C 67,8°C 68°C
- 1 % agarose gel -> 25 min, 140 V
- gel picture:


- used annealing temperature of 60,2°C (B) for preparative PCR (50 µl / reaction):
- two different reactions with different template amounts:
- reaction 1: 0,5 ng
- reaction 2: 5 ng


component volume in µl
Q5 2xMastermix 25
primer Q5 (10µM) 2,5
primer Q8 (10µM) 2,5
water reaction 1/2 19/10
template reaction 1/2) 1/10
total volume 50


- 1 % agarose gel -> 30 min, 120 V
- excision of gel bands from reaction 1 and 2 (1,3 kb) -> put in one tube
- stored at 4 °C over night


Tuesday, 08/22/17

- Gel clean-up of dCas9-A (Elution with 50 µl NE-Buffer) -> c = 17,6 ng/µl
- Fusion PCR
- changes in the PCR Program (in Torsten):
- elongation time in the second cycle: 3 min 18 s (30 s/kb * 6,6 kb (size of the fusion product) = 198 s)
- finale elongation time after the second cycle: 10 min
- for the first part of the fusion PCR:


component volume in µl
Q5 2xMastermix 12.5
dCas9-A (50 ng/µl) 2.8
dCas9-B (50 ng/µl) 1.6
water 8.1
template reaction 1/2) 1/10
total volume 25


at the "hold-step" in PCR program added the primer mix (5 µl) and continued the PCR:


component volume in µl
Q5 2xMastermix 2.5
dCas9-A (50 ng/µl) 1.25
dCas9-B (50 ng/µl) 1.25
total volume 5


- DpnI digestion after the fusion PCR:
- added 1 µl DpnI to the PCR product
- incubated 30 min at 37°C
stored digested PCR product at -20 °C


Wednesday, 08/23/17

- gel extraction of fusion-PCR product (1 % agarose, next time use 0, 7%; 100V. 55 min)
- elution in 45 uL NE-Buffer (25 + 20), concentration: 3,3 ng/µL
- not enough --> PCR amplification with Q5/Q6 with standard 50 µL reaction (0,5 µL Plasmid template)
- program "dCas9 after fusion in PeqSAR mixer, Eppi: "PCR fusion", 35 cycles

temperature time
98 30
98 10
64 30
72 3:21 min
72 2 min


- into -20, tomorrow test gel run
- Primer for amplification (Q25-Q30) resuspended to 100µM and separately diluted to 10 µM
- IDT-fragments were diluted 1:10 separately
- PCR for amplification of PCR fragments:
- pipetted normal 50 µL reaction (1 µL of diluted fragment)
- PCR reaction ("idt" in Torsten): 35 cycles


temperature time
98 30
98 10
gradient 58-71 20
72 60
72 2 min


- PP7 and MS2 at 71 °C, LacI at 58 °C
- put into -20 and tomorrow test gel run


Thursday, 08/24/17

- Fusion PCR: purifaction gel electrophoresis
- gel electrophoresis:
- 50 µl DNA
- 10 µl Loading Dye
- 0,7 % Agarose gel
- 6,6kb fragment
- 1h with 80 V
- purifaction with the Promega Purification Kit
- final concentration: 45,7 ng/µL

- test gel of PCR with IDT fragments of yesterday:
IAAH-PP7 - IAAM-MS2 - LacI - TaC1 - TaC2 - TaC3 - Ddx4-YFP


- only IAAH-PP7 and TaC3 bands are okay
- gel purification: PP7: 40,7 ng/µL
- TaC3: 27,3 ng/µL
- second PCR with other fragments, used three different temperatures for annealling for every fragment:


fragment 1st TM (well) 2nd TM 3rd TM
MS2 68,3 (9) 69,7 (10) 71 (12)
LacI 56 (1) 57,3 (3) 60,7 (5)
TaC1 66,3 (8) 68,3 (9) 69,7 (10)
TaC2 68,3 (9) 69,7 (10) 71 (12)
YFP 62,6 (6) 64,4 (7) 66,3 (8)


- Tip from Fabian: next time more gradient Temperature distance!
- fragments from left to right (Temperatures are alaso from left to right) : MS2, LacI, TaC1, TaC2, YFP
2017-08-24 15hr 44min-1.jpg
- MS2 / 9 band correct
- YFP / 6 and 7 bands are correct
- LacI no bands, TaC1 almost no bands, TaC2 bands incorrect
- gel run of MS2/9 and YFP/ 6,7 and put cut gel slices in the fridge for extraction tomorrow
- test gel of 4 uL (10 ng/uL) of IDT-fragments: LacI, TaC1, TaC2 with 6 uL H2O und 2 uL Dye to see if there's something wrong with the IDT fragments


- TaC1 and YFP show correct bands
- TaC2 correct and wrong band (maybe low concentration?) --> Nanodrop result: 11,4 ng/uL; problem that DNA concentration of desired
DNA is too low for restriction?


Friday, 08/25/17

- Gel and PCR clean up (Promega kit)
- MS2: 240mg gel; final concentration: 16,6 ng/µL
- YEP 6: 250mg gel; final concentration: 19,6 ng/µL
- YEP 7: 200mg gel; final concentration: 10,9 ng/µL
Gibbson Assembly "NEBuilder® HiFi DNA Assembly Master Mix"
https://www.neb.com/protocols/2014/11/26/nebuilder-hifi-dna-assembly-reaction-protocol


component volume in µl size in bp concentration
Vector: psB4A5 (IG_C_P1_linearized) 2,75 3395
Insert 1: IAAM-MS2 6,1 2200 10,6 ng/µl
Insert 2: IAAH-PP7 1,3 1800 40,7 ng/µl
Master Mix 10,15ul


Trafo
https://www.neb.com/protocols/2015/02/17/nebuilder-hifi-dna-assembly-chemical-transformation-protocol-e2621
- Gibbson Assembly (5 µl) + competen cells (C2987)
- plate cells: 100µl and 200µl


Monday, 08/28/17

- Gibson Assembly worked, we have colonies but they are useless (see below)
- colony PCR following the protocol (beware that the protocol has been updated in the lab),
annealing Temperature: 52°C wiht Q24 and Q18 and 10 colonies
- negative control: IG_C_P1 miniprep
- negative/Gibson 1-10 and TaC3
- negative results for all reactions except the last onw which is just the gel purified TaC3
- excpept it isnt because the band cleearlyc shwows a sifferent size and is more likely to be IAAAH-PP7 (a suspicion of Sarah who wasn't sure if the tubes were swapped) which explains the negative results in the Gibson assembly
- Gibson assembly was done again withcorrect fragments:
- pSB4A5 miniprep: 2,75 uL (18 ng/µL)
- IAAM-MS2 6,1 uL (10,6 ng/µL)
- IAAH-PP7 1,9 uL (27,3 ng/µL)
- MasterMix 10,77 µL (fragments to Mastermix : 1:1)
- two Trafos were done with already used competent cells and new competent cells (always use the cells we got from - the Gibson Kit for Gibson assembly!) and plated with 200 uL and centrifuged
- repeated PCR to amplify IDT-fragments with TaC1, TaC2 and LacI (always as 50 uL reactions):
- 3 different concentrations and temperatures for each fragment:


temperature in °C/ concentration 0,1 ng 1 ng 10 ng
60 1 4 7
68 3 6 9


from left to right: Marker- TaC1/ 1-9 - one empty lane - TaC2/1-4 - Marker Marker - TaC2/5-9 - LacI/ 1-9 (but not 5!) - Marker - 6LacI/5
low concentrations worked best, results at Tac1/1, TaC2/3 and LacI/1 were best and gel purified
- Tac1: 5,8 ng/µL
- Tac2: 9,2 ng/µL
- LacI: 12,9 ng/µL


Tuesday, 08/29/17

Colony-PCR of P4 with Q24 and Q18 (see protocol and note the changes in the folder!)
- 10 colonies
- masterplate
- did a test gel (1 % Agarose) (actualy did two beacuse of missing marker in the first one, see lab book)
- gel picture shows: all fragments have expected length
- no miniprep culturs inoculated because colonies were too small -> To do tomorrow
Gibbson assembly of P5 (IAA-RBP in pSB4A5) (see online protocol from NEB) Transformation of P5 into Jm109
- for tomorrow: inoculate miniprep cultures of positive clones on masterplate for P4, Colony-PCR and test gel for P5


Wednesday, 08/30/17

Colony PCR of P5 (low copy plasmid, see protocol) with Q19 and Q20
- gel picture: all clones okay
- inoculated three precultures for minipreps tomorrow (10 ml, 37 °C shaker)
- also inoculated three precultures of P4 for miniprep tomorrow (10 ml, 37 °C shaker)


Thursday, 08/31/17

- Miniprep of IG_C_P4 and P5 (three colonies each)

component concentration in ng/µl
P4-2 370,8
P4-4 346,1
P4-8 287,5
P5-1 153,7
P5-3 137,9
P5-8 124,7


- Eppis named like this: "IG_C_P4_2_Mini"
- test digests:
P4:


colony 2 4 8
200 ng Plasmid 0,5 µL 0,6 µl 0,7 µl
NotI-HF 1 µl 1 µl 1 µl
10 x cutSmart buffer 1 µl 1 µl 1 µl
ddH2O 7,5 µl 7,4 µl 7,3 µl
total volume 10 µl 10 µl 10 µl


P5:
digest to see if mutation worked

colony 1 3 8
200 ng Plasmid 1,3 µL 1,5 µl 1,6 µl
EcoRI-HF 1 µl 1 µl 1 µl
10 x cutSmart buffer 1 µl 1 µl 1 µl
ddH2O 6,7 µl 6,5 µl 6,4 µl
total volume 10 µl 10 µl 10 µl


test digest in general:

colony 1 3 8
200 ng Plasmid 1,3 µL 1,5 µl 1,6 µl
SpeI 1 µl 1 µl 1 µl
HindIII-HF 1 µl 1 µl 1 µl
10 x cutSmart buffer 1 µl 1 µl 1 µl
ddH2O 5,7 µl 5,5 µl 5,4 µl
total volume 10 µl 10 µl 10 µl


we used HindIII-HF and NotI-HF from the group but bought one NotI-HF again for them ran digests on 0,7 % gel:


- P5 looks good on both digests,
- P4 only one band but probably just two bands which semm ike one so it should be fine
- glycerol stock was put in -80 °C of P4 2,4 and 8 and P5 1 and P5 3


Friday, 09/01/17

- diluted P4 / 2,4,8 and P5/ 1,3 to 100 ng/µl and Q21/q22 to 5 µM
- sent fo rsequencing as follows:


component sequenze
P4-2 forward 33FE16
P4-2 reverse 33FE17
P4-4 forward 33FE18
P4-4 reverse 33FE19
P4-8 forward 33FE20
33FE21
P5-1 forward 33FE22
P5-1 reverse 33FE23
P5-3 forward 33FE24
P5-3 reverse 33FE25


Monday, 09/04/17

- prepared 2 ml over night culture for glycerol stocks
- IG_C_P4 clone 2, 4 and 8
- IG_C_P5 clone 1 and 3


Tuesday, 09/05/17

- Preparation for Gibson assembly: PCR with Q5 polymerase for P5 (to amplify LacI-dCas9 with overhangs to P4) and P4 (to open the plasmid)
- prepared dilutions of 1 ng/µl for each template:


template starting concentration in ng/µ water in µl plasmid in µl
P5 clone 1 153.7 153 1
P5 clone 3 137.9 137 1
P4 clone 4 346.1 345 1
P4 clone 8 287.3 286 1
P4-clone 2 1
P4-clone 2 370.8 370 1


- Master Mix:

component primer for P5 volume for P5 in µl primer for P4 volume for P4 in µl
Q5 Master Mix 62.5 87.5
Primer 1 Q9 6.25 Q11 8.75
Primer 2 Q10 6.25 Q12 8.75
Water 47.5 66.5
sum 122.5 171.5


-> 49 µl Master Mix + 1 µl diluted Template (1 ng/µl -> 1 ng)
- PCR programs (name in Torsten: P4_P5_PCR-vor-Gibson)
- PCR programs how they should be: - amplified fragment size
- P4: 7309 bp - P5: 5680 bp

P5 P4 cycles
98°C 30s 62.5
98°C 10s 30 6.25 30
57°C 30s 30 62°C 30s 30
72°C 170s 30 72°C 220s 30
72°C 2 min 72°C 2 min


-> made a gradient PCR to performe both PCRs simultaneously!
-> P5 in well 5 (58.1 °C)
-> P4 in well 8 (61.9 °C)
- test gel (0.7 % agarose): used 2 µl of PCR product + 8 µl water + 2 µl LD (loading dye)
- 130 V, 30 min



Wednesday, 09/06/17

- remove master plate (Gibson Assembly for P4) from waste
- repeat colony PCR, because sequencing results are negative (all clones)
- expected size: 700bp
- colony PCR: difficult to analyze (too many bands)
- repeat PCR for IAAM_MS2
- 50 µl Reaction
- 25 µl Q5 MasterMix
- 2,5 µl Primer Q27
- 2,5 µl Primer Q28
- 1 µl Template
- 19 µl ddH2O
- IDT PCR Programm
- Anealing temperature = 68,3°C
- Extension time = 66s
- Trafo of Gibson Assembly (28.08.17; P4 in C2987) https://www.neb.com/protocols/2015/02/17/nebuilder-hifi-dna-assembly-chemical-transformation-protocol-e2621
- Gibson Assembly (5 µl) + competen cells of NEB (C2987)
- plate cells: 100 µl and 200 µl
- Gel and PCR clean up (Promega kit)
- LacI-dCas9 from P4: final concentration: 114,1 ng/µL
- tried Salkowski mixture with Nitrocellulose membrane (lab 1.17 above EtBr place, orange box)
- membrane did not dissolve, so we can do this now


Thursday, 09/07/17

- PCR for IAAH-PP:
-charging stock:
component volume in µl
Mastermix (Q5 2x) 25
Q25 2.5
Q26 2.5
IAAH-PP7 1
ddH2O 19


- annealing temp. = 71°C
- extension time = 55s
- Gel run: PP7 has expected size
- c of PP7 =409,1 ng/uL
- Colony PCR of Gibson Assembly:
- charging stock:



component volume in µl
ddH2O 92,3
Q18 11.7
Q24 11.7
Taq Polymerase 144.3


- per aliquot: 20 µl
- picked 10 clones, just numer 2 was useful -> pre culture for miniprep with this one
- MS2:
- testgel with 2 µL of PCR product, 8 µL ddwater, 2 µL LD
- preparative gel
- gel clean up
- c = 10,8 ng/µL


Friday, 09/08/17

1. Redo of Gibson Assembly of IAAM-MS2 + IAAH-PP7 into psB4A5
- Gibson assembly (done with same ratios like before)
- pSB4A5 miniprep: 2,75 µL (18 ng/µL)
- IAAM-MS2 6,1 µL (10,6 ng/µL)
- IAAH-PP7 0,1 µL (409,9 ng/µL) <- only with different volume due to higher concentration (amplified DNA from 7.9)
- MasterMix 10,77 µL (fragments to Mastermix : 1:1)
- Trafo of Gibson Assembly (P4 in C2987)
- https://www.neb.com/protocols/2015/02/17/nebuilder-hifi-dna-assembly-chemical-transformation-protocol-e2621
- Gibson Assembly (5 µl) + competen cells of NEB (C2987)
- plate cells (Carb): 100 µl and 200 µl
2. Miniprep of clone 2 (see page 50 in labbook) with psB4A5: IAAM-MS2+IAAH-PP7
- used Promega miniprep kit + protocol
- concentration: 380,8 ng/µl
- made glycerol stock using 700 µl cells + 700 µl Glycerol 80 %
- Test digest of P4 (miniprep) with
- 0,53 µl DNA (miniprep)
- 0,5 µl KpnI
- 1 µl CutSmart
- 7,7 µl ddH2O
- Gel picture see lab book

Monday, 09/11/17

- pictures fare from the same gel, left looks a bit promising but with an extra band (maybe uncut plasmid?), right is with more lighting and only one band
- made colony PCR of Gibson plates from yesterday -> all 16 clones negative,
- Salkwoski was still prepared and has to be done tomorrow
- made restriction digest like yesterday to check if the correct insert is there and digest Friday was just bad but it was also negative
- Gibson assembly was done again:


component volume in µl
IG_c_P1 linearized (18 ng/µL) 2,75
IAAM-MS2 (10,6 ng/µL) 6,1
IAAH-PP7 (19,8 ng/µL) 2,7
11,55


- IaaM from the 7.9. was empty!
- IAAH-PP7 was purified (it was not purified and the unpurified was used for last Gibson so there is probably the mistake)
- incubated gibson for 40 minutes at 50°C and plated on Amp
- plated with 4 g/L TrP which was done today, too


Tuesday, 09/12/17

- Colony PCR of the Gibson Assembly (P4)
- 11 clones, all negative
- Gel Electrophoresis: 0,7 %, 120V, 25 min


temperature in °C time in sec
95 60
95 15
52 15
72 11


- 30 cycles
- Gibson Assembly
- 50 min at 50 °C
- approach
- 6,1µl IAAM-MS2 (10,6 ng/µl)
- 2,7µl IAAH-PP7 (19,8 ng/µl)
- 1,2µl ddH2O
- 10µl MasterMix


Wednesday, 09/13/17

- PCR
- approaches
1) 1 µl Gibson Assembly as template
2) 1 µl (1:10 diluted)Gibson Assembly as template
3) 1 µl (1:100 diluted)Gibson Assembly as template
- Master Mix
- 75 µl Q5 Master Mix
- 7,5 µl Primer Q26 (10µM)
- 7,5 µl Primer Q27 (10µM)
- 57 µl ddH2O
- PCR
- 49 µl Master Mix + 1 µl Template


temperature in °C time in sec
98 30
98 10
71 30
72 120
72 120
10


- 35 cycles
- Test Gel
- 2 µl PCR Product + 8µl ddH2O + 2µl loading Dye
- all negative
- second PCR
- approaches
- 9 * 1:10 diluted (tube 1-9)
- 9 * 1:100 diluted (tube 10-18)
- PCR with gradient


temperature in °C tube
60,1 1,10
60,9 2,11
61,8 3,12
63,1 4,13
64,4 5,14
65,6 6,15
66,9 7,16
68,2 8,17
69,1 9,18


Friday, 09/15/17

- prepared P5 for shipping to iGEM HQ
- used Q37/Q38
- PCR program: dCas9 iGEM
temperature in °C time in sec
98 30
98 10
65 15
72 1:50 min
72 2 min


- standard pcr protocol with 50 µL reaction and 1 ng template
- ran gel for 50 min at 90 V
- expected size 5700 bp
- gel extraction (forgot test gel so I just cut it out even if it was just one band)
- labelled epp: "IG_CP5" for iGEM HQ
- final concentration: 95,7 ng/µl
- for monday: restriction with EcoRI and PstI, ligation into pSB1C3 and Trafo


Monday, 09/18/17

- PCR for IaaH-PP7 to get rid of erroneous overhang (overhang to IaaM-MS2)
- reaction mix


component volume in µl
Q5 Master mix 25
IG_C_Q26 (10µM) 2,5
IG_C_Q39 (10µM) 2,5
IaaH-PP7 IDT (1:10) 1
water 19
sum 50


- annealing temperature: 71°C
- elongation time: 54 s
- expected fragment size: 1,8 kb
- Gel clean-up of PCR product
- 0.7 % agarose gel -> run 1 h at 80 V
- cut the band at ~ 1,8 kb
- purification of fragment with promega kit
- concentration of IaaH-PP7: 7,8 ng/µl
- Gibson assembly for P4


component volume in µl
Assembly Master mix 15.65
IaaM-MS2 (10.6 ng/µl)
IG_C_Q39 (10µM)IaaH-PP7 (7.8 ng/µl) 6.8
pSB4A5 (IG_C_P1 linearized, 18 ng/µl) 2.75
sum 31.3


- incubation: 63 min at 50 °C
- transformation of 5 µl assembly mix into 50 µl competent DH5a (C2987)
- plated on amp + trp plates (200 µl and rest)
- digestion of PCR product IG_C_P5 for iGEM HQ (from 15.09., LacI-dCas9) with EcoRI and PstI


component volume in µl
Cutsmart 1
EcoRI 1.5
PstI 1.5
LacI-dCas9 (400 ng) 4.18
water 1.82
sum 10


- incubation: 2h 10 min at 37 °C
- Gel clean-up of digestion product
- 0.7 % agarose gel -> run 1 h at 80 V
- cut the band at ~ 5700 kb
- purification of fragment with promega kit
- concentration of digested LacI-dCas9: 10.2 ng/µl
- Ligation of purified and digested LacI-dCas9 into pSB1C3 (digested with E + P, 28.08.)


component volume in µl
T4 DNA Ligation Buffer 2.5
1
LacI-dCas9 (10.2 ng/µl) 10.7
pSB1C3 (12.5 ng/µl) 8
water 2.8
sum 25


- incubation: 1 h 8 min at room temperature
- transformation of 5µl ligation mix into 50 µl competent DH5a (C2987)
- plated on cap plates (200 µl and rest)


Tuesday, 09/19/17

- Colony PCR of dCas9-LacI in pSB1C3
- mastermix for 10 clones + negative control


component volume in µl
water 100,8
Q17 9,6 -> annealing Tm 52°C, binds to backbone
Q20 9,6 -> annealing Tm 52,6°C, binds to dCas9
Red Taq 120


- fragment length: 1,6 kb -> elongation time 23 sek
- gel picture: except for clone 3 and 9 all clones positive
- inoculated miniprep cultures (5 ml LB + 5 µl CAP) of clone 4,7,8 and 10
- Colony PCR of P4
- mastermix for 10 clones + negative control


- Colony PCR of dCas9-LacI in pSB1C3
- mastermix for 10 clones + negative control


component volume in µl
water 100,8
Q18 9,6
Q24 9,6
Red Taq 120


- annealing Tm 52°C
- fragment length: 700 bp -> elongation time: 10,5 sek
- gel picture: no bands!
- possible reasons: not enough DNA (IAAH-PP7 with corrected overhangs) applied due to low concentration (see 18.09.) or colony PCR did not work properly. In case of that miniprep cultures were inoculated with clone 1,6,8 and 10 (10 ml LB + 10 µl Amp)
- Tomorrow test digest of those!
- master plate with cellulose membrane in 37 °C incubator
- Amplification of IAAH-PP7 with corrected overhangs
- gradient PCR: tried 1:10 and 1:100 dilutions
- master mix for 11 x 10 µl aliquots


component volume in µl
water 48
Q39 8
Q26 8
Q5 master mix 80


- per tube: 9 µl master mix + 1 µl template (1:10, 1:100 or 1:1000)
- same gradient, same conditions
- gel: 1% agarose; 150 V, 30 min
- clear bands visible, all clones positive!
- annealing temperatures between 65-67 °C (all three equaly good) and concentration of 1:1000
( 0,01 ng/µl) worked best
- extracted all bands at 1,8 kb from gel -> three yellow labeled tubes in the fridge